This proposal combines the efforts of researchers in the Departments of Chemistry and Pathology at the University of Washington and at the Fred Hutchinson Cancer Research Center. These researchers will perform a pilot study to evaluate new technology for cancer research. This technology produces protein fingerprints from single cells and will be evaluated for the study of cells isolated from biopsies obtained from patients in the Seattle Barrett's Esophagus Cohort. This Application of Innovative Technologies for the Molecular Analysis of Cancer proposal is the first study of cell-to-cell variation in protein expression in human precancerous tissues. In the R21 phase of this project, we will demonstrate the generation of a two-dimensional capillary electrophoresis fingerprint of the proteins from single cells isolated from an esophagus tissue biopsy. Once we demonstrate that we can successfully analyze a single cell isolated from the solid tissue, we will proceed to the R33 phase of the project. During that phase, we will perform several experiments of interest to cancer biologists. We will dissect and generate two-dimensional protein fingerprints from 20 cells from a single crypt isolated from a biopsy. This study will provide understanding of the change in protein expression during development of a stem cell into its differentiated form. We will generate fingerprints from three crypts obtained from a single biopsy. These crypts will be closely related and the study will provide information of the intraclonal variation in protein expression. We will generate fingerprints from cells isolated from crypts obtained from three biopsies from the same patient. This study will provide information on the interclonal variation in protein expression. We will generate fingerprints from 10 patients, 5 negative for dysplasia and 5 with high grade dysplasia. This study will provide information on the differences in protein expression between patients at low risk and patients at high risk for development of esophageal adenocarcinoma.